The art of organic chemistry on solid supports is generally known. A useful review article on this topic may be found in "Organic Chemistry on Solid Supports" by Fruchtel et al., Angew. Chem. Int. Ed. Engl., 1996, 35, pgs. 17-42, the contents of which are hereby incorporated by reference.
As discussed in Fruchtel et al., the art has developed automated solid-phase synthesis of polypeptides, oligonucleotides and oligosaccharaides. Of particular interest here is solid-phase synthesis of oligonucleotides. The following are useful review articles/textbooks on this topic:
Beaucage et al., Tetrahedron, 1992, 48, 2223; PA1 Davis et al., Innovation and Perspectives in Solid Phase Synthesis (Ed.: R. Epton), Intercept, Andover, 1992, pg. 63; PA1 Montserra et al., Tetrahedron, 1994, 50, 2617; and PA1 S. L. Beaucage et al., Tetrahedron, 1993, 49, 6123-6194; PA1 Caruthers et al., Genetic Engineering, Plenum Press, New York (1982), Vol. 4, pgs. 1-17; PA1 Letsinger et al., Genetic Engineering, Plenum Press, New York (1985), Vol. 5, pg. 191; PA1 Froehler et al., Nucleic Acids Research, 14:5399-5407 (1986); and PA1 Matteucci et al., Journal of American Chemical Society, 103:3185-3186 (1981);
the contents of each of which are hereby incorporated by reference.
In the solid-phase synthesis of oligonucleotides, it is known to synthesize the oligonucleotide on an inorganic solid support bearing a succinyl linker arm--see, for example, any of the following references:
the contents of each of which are hereby incorporated by reference.
Typically, the succinyl linker arm has the following general formula: ##STR1## Thus, the succinyl group links the growing oligonucleotide from its terminal 3' hydroxyl group by an ester bond to a primary amine on the support, which may be, for example, conventional controlled pore glass (CPG) or silica, by an amide bond. Once the desired oligonucleotide has been synthesized, it is freed or cleaved from the succinyl linker arm hydrolyzing the ester carbonyl group. The hydrolysis agent is usually concentrated ammonium hydroxide. Typically, this reaction can take from 1-4 hours to complete. With improvements to current solid-phase oligonucleotide synthesizers, this cleavage step can represent 50% or more of the total time require to synthesize the desired oligonucleotide.
Another type of linker arm is disclosed in U.S. Pat. No. 5,112,962 [Letsinger et al. (Letsinger)], the contents of which are hereby incorporated by reference. Letsinger teaches a linker arm for solid support synthesis of oligonucleotides and oligonucleotide derivatives have the following formula: ##STR2## Thus, Letsinger teaches an oxalyl linker arm which purportedly release the synthesized oligonucleotide or oligonucleotide derivate in a period of 1-30 minutes in a manner that leaves the oligonucleotide fully protected. The oxalyl linker arm purportedly can be rapidly cleaved by 5% ammonium hydroxide in methanol, ammonium hydroxide, wet tertiary amine, triethylamine/alcohol, triethylamine/methanol, triethylamine/ethanol, aqueous trimethylamine and other bases. Unfortunately, the oxalyl linker arm of Letsinger suffers from its purported advantage. Specifically, the present inventors have discovered that the oxalyl linker arm of Letsinger is susceptible to significant spontaneous hydrolysis (e.g. spontaneous hydrolysis of .about.10-40% per month) which renders it difficult to use in commercial operations. The oxalyl arm is also difficult to prepare because it requires using oxalyl chloride, which is highly reactive, toxic and therefore dangerous.
Regardless of the specific nature of the linker arm, it is generally accepted in the art that the linker arm is not reusable after production and cleavage of the desired oligonucleotide. Thus, conventional linker arms may be regarded as non-recyclable. This is illustrated in FIG. 1 which illustrates the conventional use of a succinyl linker arm for the production of an oligonucleotide. Thus, as illustrated, after cleavage of the desired oligonucleotide, the support is irreversibly linked to the linker compound (i.e. the succinyl moiety) and cannot be reused.
The art is in need of a linker arm for solid support oligonucleotide synthesis, which linker arm is recyclable. More specifically, the art is in need of a linker arm capable of repeated oligonucleotide synthesis/cleavage.